Abstract Tuberculosis remains a major global health threat, as Bacillus Calmette–Guérin (BCG), the only licensed vaccine, provides limited protection, particularly in adolescents and adults. To address this limitation, a more effective tuberculosis vaccine was developed using the SpyTag/SpyCatcher system to display five clinically validated Mycobacterium tuberculosis antigens (Ag85A, ESAT-6, CFP10, Rv2660c, and TB10.4) on self-assembling mi3 nanoparticles. These nanoparticle-displayed antigens, formulated as 85A-NP, EC-NP, and RT-NP and combined with a custom AS01E-biosimilar adjuvant, elicited stronger Th1-biased immune responses in C57BL/6 mice than the corresponding recombinant proteins, as evidenced by increased frequencies of polyfunctional CD4⁺ T cells producing IFN-γ, IL-2, and TNF-α. In a murine aerosol challenge model, the mixed nanoparticles formulation (85A-NP:EC-NP:RT-NP) conferred superior pulmonary protection compared to single-antigen nanoparticles, recombinant protein mixtures, an in-house M72-like vaccine and BCG. This modular platform enables efficient multistage antigen incorporation and holds promise for next-generation tuberculosis vaccine development.
